Multi-drug resistance is one of the most pressing issues in treating bacterial infections. Antibiotics are extruded from cells and cannot reach high enough intracellular concentrations to exert a therapeutic effect. This problem is most formidable with Gram-negative (Gram (-)) bacteria, due to their double-membrane structure. While efforts have focused on targeting one efflux pump at a time, resistance mutations can quickly develop. We propose to target the m1G37-tRNA methylation catalyzed by TrmD to inhibit protein synthesis of multiple pumps simultaneously, thus reducing drug efflux and accelerating bactericidal action. TrmD is a bacteria-specific S-adenosyl-methionine (AdoMet)-dependent methyl transferase that controls the accuracy of protein-synthesis reading frame. Loss of TrmD increases +1 frameshifts and terminates protein synthesis prematurely. We have discovered that genes for multiple membrane proteins and efflux pumps in E. coli and other Gram (-) bacteria contain TrmD-dependent codons near the start of the reading frame. We hypothesize that targeting TrmD will reduce protein synthesis of all of these genes. By reducing multiple membrane and efflux proteins at once, we propose that targeting TrmD offers a novel solution to an unmet medical need. While AstraZeneca (AZ) has attempted to target TrmD, progress has stalled, because isolated inhibitors lacked both the selectivity against the human counterpart (Trm5) and the activity against bacterial growth. We hypothesize that successful targeting must explore novel chemical space and diversity to capture the unique conformation of AdoMet when bound to TrmD. To test this hypothesis, our multi-PI team will use E. coli TrmD (EcTrmD) as a model and apply a series of high-throughput screening (HTS) assays, each unique to our team, to isolate potent and selective inhibitors. In Aim 1, we will use an enzyme-based fluorescence assay to isolate active inhibitors of EcTrmD. This fluorescence assay is HTS-ready, has all of the required reagents in hand, and exhibits advantages over the radioactivity-based (3H-AdoMet) assay. We will screen the collection of ~370,000 compounds in the NCATS SMR (small molecular repository) at Sanford Burnham Prebys (SBP) and will apply human Trm5 in a counter screen to remove non-selective compounds. In Aim 2, we will use cheminformatics to prioritize hits. We will assess hits in a multitude of secondary assays to determine their inhibition potency and modality. In Aim 3, we will screen hits with our whole-cell assays to isolate compounds that inhibit cell growth and display phenotypes specific to TrmD deficiency, including reduced drug efflux. We will assess the structure-activity relationship of each hit by analysis of ~20 analogs from commercial vendors and determine the binding modality using a computer-aided approach based on our ternary TrmD crystal structure in complex with a bound tRNA and sinefungin (a non-reactive analog of AdoMet). We will determine hits for specificity of targeting EcTrmD inside E. coli cells. These hits will serve as powerful chemical probes in a new paradigm of antibiotic discovery that inhibits Gram (-) bacterial drug efflux by targeting TrmD.